Minimally-invasive measurement of esophageal inflammation

ABSTRACT

The methods and apparatus of the present invention allow the evaluation of inflammation of the esophagus. Measurements may be utilized, for example, to diagnose a disease of the esophagus, to monitor inflammation of the esophagus, or to access the treatment of a disease of the esophagus. In one embodiment, the invention comprises a method for measuring esophageal inflammation comprising deploying a device into the esophagus of a subject, removing the device after a predetermined period of time, analyzing the device for a diagnostic indicator of esophageal inflammation and evaluating the diagnostic indicator to diagnose esophageal inflammation.

This application claims the benefit of U.S. Provisional Application No.60/985,386, filed Nov. 5, 2007, the contents of which are herebyincorporated by reference.

This invention was made with government support under grant numbersR21-AI079925 awarded by the National Institutes of Health (NIH). Thegovernment has certain rights in the invention.

BACKGROUND OF THE INVENTION

A. Field of the Invention

The present invention relates generally to the field of medicine. Moreparticularly, it concerns methods of diagnosing and monitoring diseasesof the esophagus.

B. Description of Related Art

Making the diagnosis of many inflammatory conditions of thegastrointestinal tract such as severe gastroesophageal reflux (GERD),eosinophilic gastroenteritis (EGE), food allergic enteropathy (FAE), andinflammatory bowel disease (IBD) is often difficult. Serologic andradiographic assessments are not diagnostic for any of these conditions.In the instance of food allergic disorders, skin prick testing and RASTanalysis are the only non-invasive tests available. In addition, thesetests are beneficial only in identifying IgE mediated reactions and arenot useful for cell-mediated reactions. In other instances, non-invasiveaccess to the gastrointestinal (GI) tract is only available throughradiographic or stool analysis and again these do not provide definitivediagnostic information for these disorders.

Ultimately, endoscopic analysis is required to obtain mucosal samplingfor diagnosis and to develop a treatment plan. Affected tissue sectionsare characterized by infiltration of polymorphonuclear leukocytes andeosinophils into the diseased sites. For some diseases, such as Crohn'sdisease, histopathological features are clear and characterized by thefinding of chronic ileal inflammation with non-caseating mucosalgranulomas. For other diseases, such as eosinophilic gastroenteritis andfood allergic disorders, the exact histological features are not ascertain and a degree of overlap with other conditions exists.

Methods to study esophageal diseases are limited to in vitro models, afew animal models and human studies of esophageal biopsies. Other,“minimally-invasive” esophageal studies can measure the amount of acid(pH monitor), non-acid (impedance monitor), and bile (Bilitec monitor)in the esophageal lumen. These later tests require placement of an inertprobe (approximately 3 mm diameter) through the nose and into the distalesophagus for overnight monitoring. Another test of esophagealinflammation involves swallowing a capsule with a camera that can takepictures of the esophagus. Finally, esophageal function can be measuredby placing probes into the esophagus for a pressure and waveformmeasurements (manometry/motility monitors).

U.S. Pat. No. 6,475,145 discloses a method utilizing a retrievable andingestible radioactive capsule swallowed by a subject and held in placeby means of a cord for diagnosis of GERD and acid-reflux. Theradioactive capsule is degraded below pH4 so the measure is aradioactive release. The cord provides a means for retrieving thecapsule.

U.S. Pat. No. 5,738,110 describes a device for the diagnosis of certaingastrointestinal pathogens. This device comprises a gelatin capsulewhich contains a drag material which has thin sample cloth embeddedtherein, which is attached to a string. The patient holds the string andswallows the capsule, allowing the sample cloth to come into contactwith the small intestine. After a period of time, the sampling cloth andthe drag material are recovered for testing.

To date, no method has been used to quantify the inflammatory contentsof the esophageal lumen. Analogous to bronchial lavage in pulmonarydiseases, stool collections in intestinal diseases and urinalysis inrenal diseases, measurement of esophageal contents allows a directdefinition and measurement of inflammatory mediators associated withesophageal diseases such as GERD and EE. A safe, “minimally invasive”alternative to endoscopy with biopsy to assess esophageal inflammationis therefore needed.

SUMMARY OF THE INVENTION

The methods and apparatus of the present invention allow the evaluationof inflammation of the esophagus, by example, for diagnosis of diseaseof and assessment of treatment for a disease of the esophagus. In oneembodiment, the invention comprises a method for measuring esophagealinflammation comprising deploying a device into the esophagus of asubject, removing the device after a predetermined period of time,analyzing the device for a diagnostic indicator of esophagealinflammation and evaluating the diagnostic indicator to diagnoseesophageal inflammation.

In another embodiment, the present invention provides a method fordiagnosing a disease of the esophagus comprising deploying a device intothe esophagus of a subject, removing the device after a predeterminedperiod of time, analyzing the device for a diagnostic indicator of adisease of the esophagus and evaluating the diagnostic indicator todiagnose a disease of the esophagus.

In other embodiments, the invention comprises a method for assessing atreatment of a disease of the esophagus comprising deploying a deviceinto the esophagus of a subject, removing the device after apredetermined period of time, analyzing the device for a diagnosticindicator of a disease of the esophagus and evaluating the diagnosticindicator to assess the treatment of a disease of the esophagus.

The current invention may be used to measure any cause of inflammationof the esophagus. This measurement may be utilized, for example, todiagnose a disease of the esophagus, to monitor inflammation of theesophagus, or to access the treatment of a disease of the esophagus. Thedisease of the esophagus may comprise inflammation of the esophagus. Forexample, the current invention may be used to assess and diagnosegastroesophageal reflux disease (GERD) or complications associated withGERD such as Barrett's esophagus or cancer. In another non-limitingembodiment, the disease may be Eosinophilic Esophagitis (EE).

The device may be any apparatus which allows the capture of theinflammatory mediators and other cells of the esophagus. In oneembodiment, the device comprises a pharmaceutical capsule having anopening, a drag material within the capsule, and a line embedded in thedrag material that runs through the opening of the capsule. Thepharmaceutical capsule may be dissolvable or it may pass through thesubject's system. In another embodiment, the capsule may be comprised oftwo parts, a base and a cap, and the opening of the capsule may be aperforated opening. The drag material may be constructed of variousmaterials. In some embodiments, the drag material is malleable. The linemay be of various lengths and may be made of various materials to bemore or less abrasive and more or less absorptive. In a particularembodiment, the line comprises two components, a string and a samplingcloth. The string component may be referred to as the proximal segment,whereas the sampling cloth may be referred to as the distal segment. Insome embodiments, after deployment of the string, the proximal segmentis located in the esophagus of the subject. The string may be made of,for example, an absorbent, mesh, or textured fiber. In one embodiment,the string and sampling cloth are constructed of the same material. Insome embodiments, a portion of the string may be pulled out of thecapsule prior to swallowing the capsule and the end of the string may beattached to the cheek. After the predetermined period of time, thestring may be removed from the esophagus.

In another embodiment, the device further comprises a capture agent forone or more diagnostic indicators. These capture agents may be locatedon the line. In a particular embodiment, the capture agents may befocused on the proximal segment of the line. The capture agent may beany agent that binds an analyte through an interaction that issufficient to permit the agent to bind and concentrate the analyte froma homogeneous mixture of different analytes. The binding interaction maybe mediated by an affinity region of the capture agent. Representativecapture agents include antibodies, and more specifically monoclonalantibodies. Further non-limiting examples include eosinophil granuleprotein antibodies.

The predetermined period of time may be various lengths of time. Forexample, the predetermined time may be between 15 minutes and 12 hours.In some embodiments, the predetermined period of time may be 1, 2, 3, 4,5, 6, 7, 8, 9, 10, 11, 12, 18, 24 or more hours, or any incrementthereof. In one embodiment, the predetermined period of time is 15minutes. In another embodiment, the predetermined period of time is 1hour. In a further particular embodiment, the predetermined period oftime is 12 hours.

Following removal of the device from the esophagus, the device isanalyzed for diagnostic indicators. The line may be made of variousmaterials to be more or less abrasive and more or less absorptive. Insome embodiments, the indicator is adsorbed in the device. In otherembodiments, the indicator binds to the outside of the device. In yet afurther embodiment, liquid may adhere to the device. In yet a furtherembodiment, secretions may adhere to the device. As noted above, in oneembodiment a capture agent for one or more diagnostic indicators may bepresent on the device, wherein the diagnostic indicator may bind to thecapture agent. Secretions and cells can be examined by a number ofdifferent techniques known in the art. The presence of inflammatoryproteins, RNA or cells can be analyzed within the contents removed fromthe string.

The device is analyzed for a diagnostic indicator of esophagealinflammation. In some embodiments, the device is analyzed for thepresence of one or more diagnostic indicators. In other embodiments, thedevice is analyzed for the level of one or more diagnostic indicators.Analysis may be performed by a number of methods, including but notlimited to ELISA, cytology, mass spectrometry, gas chromatography,Western Blot, Mesoscale, Licor, RNA and DNA extraction,immunohistochemical analysis, and microbial culture and staining.

The diagnostic indicator may be any factor that indicates the presenceor severity of inflammation of the esophagus. In some aspects, thediagnostic indicator may be an eosinophil granule protein, includingmajor basic protein (MBP), an eosinophil cationic protein (ECP), aneosinophil peroxidase (EPO), or an eosinophil-derived neurotoxin (EDN).In some embodiments, the diagnostic indicator is a cytokine orchemokine, such as eotaxin. In another embodiment, the diagnosticindicator is a cellular infiltrate or pH. In yet another embodiment, thediagnostic indicator is a marker of an allergic response, such as IgE,tryptase, receptor molecules (for example, FcRI or CD23) or an allergen.Other inflammatory markers that may be examined may include, forexample, arachadonic acid products and neurotransmitters such assubstance P and bradykinin. Other diagnostic indicators includeperipheral and plasma eosinophil counts, mast cells, includingleukotrienes. In other aspects, diagnostic indicator comprises one, twoor more markers.

In some embodiments, the eosinophil granule protein may be induced. In aparticular embodiment, the eosinophil granule protein is IL-5 induced.In further embodiments, the IL-5 induced eosinophil granule protein isEPO, MBP1, or CLC/Gal-10.

The diagnostic markers are evaluated to diagnose a disease of theesophagus. Evaluation may include assessment for the presence or absenceof an indicator that allows for diagnosis of a disease. For example, ifa sample shows evidence of an inflammatory reaction, this may indicatethe presence of eosinophilic esophagitis. Alternatively, the level of anindicator may be evaluated to either diagnosis or evaluate the level ofseverity of a disease. For instance, the evidence may be increasedlevels of eosinophil granule proteins that may indicate the presence ofEE. In other non-limiting embodiments, increases in specific cytokines,such as IL-6, indicates the presence of GERD. In a particularembodiment, the diagnostic indicator for GERD may be IL-8 mRNA or IL-8protein.

In one embodiment, the invention provides a method for measuringesophageal inflammation comprising deploying a device into the esophagusof a subject, removing the device after a predetermined period of time,analyzing the device for a diagnostic indicator of esophagealinflammation, and evaluating the diagnostic indicator to diagnoseesophageal inflammation. In a further embodiment, the method may furthercomprise quantifying the diagnostic indicator. The quantification may beperformed by any method known to those of skill in the art. In aparticular embodiment, the quantification is performed by ELISA. In afurther embodiment, the quantification is performed by Mesoscale.

It is contemplated that any method or composition described herein canbe implemented with respect to any other method or composition describedherein.

The use of the term “or” in the claims is used to mean “and/or” unlessexplicitly indicated to refer to alternatives only or the alternativesare mutually exclusive, although the disclosure supports a definitionthat refers to only alternatives and “and/or.”

Throughout this application, the term “about” is used to indicate that avalue includes the standard deviation of error for the device or methodbeing employed to determine the value.

Following long-standing patent law, the words “a” and “an,” when used inconjunction with the word “comprising” in the claims or specification,denotes one or more, unless specifically noted.

Other objects, features and advantages of the present invention willbecome apparent from the following detailed description. It should beunderstood, however, that the detailed description and the specificexamples, while indicating specific embodiments of the invention, aregiven by way of illustration only, since various changes andmodifications within the spirit and scope of the invention will becomeapparent to those skilled in the art from this detailed description.

BRIEF DESCRIPTION OF THE DRAWINGS

The following drawings form part of the present specification and areincluded to further demonstrate certain aspects of the presentinvention. The invention may be better understood by reference to one ormore of these drawings in combination with the detailed description ofspecific embodiments presented herein.

FIG. 1. Enterotest string test—the 90 cm nylon string is shown partiallyunraveled from the gelatin capsule.

FIGS. 2A-B. Enterotest string test. (FIG. 2A) The extended Enterotest™string is shown with the Gelatin capsule at the distal end (bottom leftarrow). The proximal end that gets taped to the patient's cheek isthinner (top right, arrow). (FIG. 2B) Segment of an Enterotest™ stringfrom a normal subject is shown with pH indicators (and indicator stick),demonstrating the location of the sections of string that were in theesophagus (alkaline pH, blue-green) and stomach (acidic pH, orange).Only the colored portions of the string were marked for pH using theindicator stick.

FIG. 3. Microbiota in the esophagus and mouth of two subjects. Totalcolonies isolated from strings resident in esophagus or mouth for 15minutes. Secondary unidentified anaerobe cultured on CDC Blood Agar withPEA not counted. No Enterococcus detected. Bars are SEM derived fromduplicate plates from single string incubated for 5 minutes in HBSS.

FIGS. 4A-B. Microbiota in the esophagus, nose, and mouth from 2subjects.

FIG. 5. PCR amplification of esophageal bacterial DNA. 16S rDNA gene.Lane 1-negative control sigma H₂O. Lane 2—40 μl bacterial DNA fromstring (S). Lane 3—40 μl bacterial DNA from string (Z). Lane 4—10 μlbacterial DNA from string (S). Lane 5—10 μl bacterial DNA from string(Z). Lane 6—1 μl bacterial DNA from string (S). Lane 7—1 μl bacterialDNA from string (Z). S DNA concentration of 1.32 ng/μl in 200 μl. Z DNAconcentration of 2.70 ng/μl in 200 μl. 40 cycles performed at 95° C. for3 min, 94° C. for 30 sec, 56° C. for 30 sec, 70° C. for 60 sec, and 72°C. for 6 min.

FIGS. 6A-F. (FIG. 6A) Endoscopic view of the esophagus of an EE patient,showing multiple eosinophil microabscesses (white dots, arrows) paintingthe inflamed epithelial surface. (FIGS. 6B-C). H&E sections showing oneof these eosinophil microabscess erupting through the epithelium intothe lumen 0, and one just beneath the surface (FIG. 6C arrow). (FIGS.6D-F). Three biopsies from the same EE patient showing normal-appearingsqaumous epithelium (FIG. 6D), a site with hyperplastic epithelium with1 eosinophil/HPF (FIG. 6E arrow), and a site with hyperplasticepithelium with >15 eosiriophils/HPF (FIG. 6F arrows), emphasizing thediscontinuity of esophageal eosinophilic inflammation in EE.

FIG. 7. Detection of eosinophil granule major basic protein-1 (MBP1)adsorbed to EST strings incubated with acidic lysates of bloodeosinophils. Acidic sonicates of purified blood eosinophils wereprepared by sonicating 1×10⁶ or 1×10⁶ cells in either 25 mM sodiumacetate/acetic acid (NaOAc) buffer (pH 4.3) or 0.1N HCI (pH 3). The ESTstrings (2 cm lengths) were incubated with 0.5 ml eosinophil sonicatesfor 1 hr, the strings removed, blotted to remove excess fluid, boiled inSDS-PAGE sample buffer, and analyzed by SDS-PAGE and Western blottingfor the presence of MBP 1 adsorbed to the EST string. Purified MBP 1 wasused as the positive control for Western blotting.

FIG. 8. Time course for detection of eosinophil peroxidase (EPO) andgranule major basic protein-1 (MBP1) adsorption to EST strings.Incubated with sonicates of blood eosinophils. Lysates of purified bloodeosinophils were prepared by sonication of 1×106 eosinophils in either25 mM sodium acetate/acetic acid (NaOAc) buffer (pH 4.3) or inphosphate-buffered saline (PBS; pH 7.3). EST strings (2 cm lengths) wereincubated with 0.5 ml of eosinophil sonicate for the indicated timepoints (1-16 hrs), the strings harvested, blotted to removed excessfluid, boiled in SDS-PAGE sample buffer, and analyzed by SDS-PAGE andWestern blotting for the presence of EPO and MBP 1 adsorbed to thestrings. The upper two panels from the same gel show both the high (˜52kD) and low (141<d) molecular weight EPO subunits, and the lower panel,MBP1. EPO and MBP1 were detected on the EST strings within 1 hr, withsomewhat more protein detected using PBS than NaOAc buffer sonicate.Incubation times longer than 1 hr did not increase the amount ofadsorbed protein detected by Western blotting.

FIG. 9. IL-5 activation of blood eosinophils results in increasedeosinophil adherence and detection of eosinophil granule cationicproteins adsorbed to EST strings. Purified blood eosinophils (1×10⁶cells) were incubated with 2 cm lengths of EST string for 1 hr incomplete culture media containing 8% FBS with or without the addition ofIL-5 (25 ng/ml) to activate eosinophil secretion. Strings were removedfrom the culture media, blotted to remove excess media, and some stringswere briefly incubated in trypsin/EDTA to remove adherent intacteosinophils. Strings were then boiled in SDS-PAGE sample buffer andanalyzed by SDS-PAGE/Western blotting for eosinophil peroxidase (EPO)and granule major basic protein-1 (MBP1) (left). Both MBP1 and the high(52 kD) and low (14 kD) molecular weight subunits of EPO are shown(left). Activation of eosinophils with 1L-5 increased the amount of MBP1and EPO detected on the EST strings (right), some of which was due toadherent intact eosinophlis that were removed by the trypsin/EDTAtreatment.

FIG. 10. Time course for detection of eosinophil granule proteinsadsorbed to EST strings incubated with IL-5 activated blood eosinophils.Blood eosinophils (1×10⁶ eos @>99%) purified from normal subjects wereactivated with IL-5 (25 ng/ml) in the presence of EST strings (2.0cm/time point) for the indicated time course in RPMI 1640 tissue culturemedia containing 8% FBS. Strings were transferred to SDS-PAGE samplebuffer, boiled and analyzed by SDS-PAGE/Western blotting usingantibodies to eosinophil peroxidase (EPO), major basic protein-1 (MBP1)and Charcot-Leyden crystal protein/Galectin-10 (CLC/Gal-10). Only thelow molecular weight subunit of EPO is shown. Positive controls forWestern blotting Included purified human EPO and MBP1, and forCLCgal-10, a lysate of AML14.3D10 eosinophils.

FIG. 11. EST detection of eosinophil derived neurotoxin (EDN) secretionby IL-5 activated eosinophils in vitro: measurement by EDN ELISA. Bloodeosinophils (1×10⁴-1×10⁶ cells @>99%) purified from a normal subjectwere cultured with (+IL-5; 25 ng/ml)) or without IL-5 (−IL-5) in thepresence of EST string (2.0 cm) for 1 hour in RPMI 1640 tissue culturemedia containing 8% FBS. Strings were transferred to 0.5% NP-40 elutionbuffer (pH 6.0) for 30 min. to elute secreted eosinophil granuleproteins, and the amount of secreted EDN analyzed by ELISA (MBL Co.,Ltd).

FIGS. 12A-B. IL-8 protein levels determined by the Enterotest in apatient with well-defined GERD. (FIG. 12A) H&E section of biopsy frompatient with GERD showing evidence of mild eosinophilic inflammation(arrow) and basal zone hyperplasia consistent with GERD. (FIG. 12B) TheEnterotest was performed in this patient and removed after 12 hours. Thestring was cut into oral, esophagus (proximal and distal), stomach andduodenum segments based on position (length) and pH indicatormeasurements. Protein secretions were eluted from the string segments,IL-8 protein levels measured using the Quantitative Mesoscale assay, andresults expressed as pg IL-8/ml/cm of the Enterotest string samples.

FIG. 13. Time course for 1L-8 protein measurement by EST in a patientwith GERD. Two separate ESTs were performed in a well-defined patientwith GERD. The strings were removed after 12 hours and 1 hour periods.Upon removal, the esophageal segment of the EST was cut into proximaland distal halves and the adherent secretions eluted with sample buffer.Secretions were assayed by Mesoscale for IL-8 protein content, expressedas pg IL-8/ml/cm of eluted string.

FIG. 14. Increased IL-8 compared to other inflammatory cytokines asdetermined by EST in a patient with GERD. EST was performed in awell-defined patient with GERD and the string removed after 12 hours.The esophageal string segment (based on pH probe and lengthmeasurements) was cut into proximal and distal halves, the adherentsecretions eluted with sample buffer, and secretions assayed byMesoscale for pro-inflammatory cytokines (protein). Note: IL-8 levels inthis figure are higher since cytokine levels are reported as pg/mlinstead of pg/ml/cm of string as in other figures.

FIG. 15. IL-8 mRNA levels determined by Esophageal String Test (EST) ina patient with GERD. The EST was performed in a well-defined patientwith GERD and the string removed after 1 hour, The string was cut intoproximal and distal esophageal sections and total RNA isolated from theadherent cells. Quantitative RT-Q-PCR was performed using IL-8-specificprimers and the results normalized to the expression of β-actin in thesamples.

FIG. 16. IL-8 protein levels measured by EST in patient with GERD beforeand after treatment. The EST was performed in a well-defined patientwith GERD before and after treatment with a proton pump inhibitor (PPI).The EST string was removed after 1 hour, the esophageal segmentidentified by pH and length and out into proximal and distal halves, andsecretions eluted and assayed by Mesoscale for IL-8 protein. Results areexpressed as pg/ml IL-8/cm eluted string

DESCRIPTION OF ILLUSTRATIVE EMBODIMENTS A. The Present Invention

The methods and apparatus of the present invention allow the evaluationof inflammation of the esophagus. In one embodiment, the inventioncomprises a method for measuring esophageal inflammation comprisingdeploying a device into the esophagus of a subject, removing the deviceafter a predetermined period of time, analyzing the device for adiagnostic indicator of esophageal inflammation and evaluating thediagnostic indicator to diagnose esophageal inflammation.

This method provides a minimally invasive method for assessing anddiagnosing a disease of the esophagus. The methods of the presentinvention may be used, for example, in assessing inflammation of theesophagus such as gastroesophageal reflux disease (GERD) andEosinophilic Esophagitis (EE). For a long period of time, eosinophilicinflammation of the esophagus was felt to be almost exclusively due toGERD. Clinicopathological analysis has now demonstrated that esophagealeosinophilia can also be due to EE, which is a disease typically havingan allergic etiology. EE has emerged as a distinct type of esophagitis,however the distinction between EE and GERD is frequently difficult toidentify. For example, the two diseases can be difficult todifferentiate by conventional methods, as GERD and EE can have similarpresentations and upper gastrointestinal series are insensitive testsfor the diagnosis of GERD. EE is characterized both by its associationwith food allergens and also by the large number of eosinophils that areusually present in the esophagus of diagnosed patients. However, in somebiopsies the eosinophil count falls within a borderline range andinflammation can be inconsistent. Eosinophils contain a number ofcytokines, chemokines, and granular proteins. These biologically activemediators are released upon activation to participate in theinflammatory cascade. The method of the current invention allows for aminimally-invasive alternative for diagnosis of these and otherdiseases.

1. Devices

The device may be any apparatus which allows the capture of theinflammatory mediators and other cells of the esophagus. In a particularembodiment, the device of the current invention comprises apharmaceutical capsule having an opening, a drag material within thecapsule, and a line embedded in the drag material and running throughthe opening of the capsule. The phrase “pharmaceutical” refers tomolecular entities and compositions that do not produce an adverse,allergic, or other untoward reaction when administered to an animal, orhuman, as appropriate. The pharmaceutical capsule may be dissolvable orit may pass through the subject's system. In some aspects, the openingof the capsule may be a perforated opening. In another embodiment, thecapsule may be comprised of two parts, a base and a cap. The dragmaterial may be constructed of various materials. In some embodiments,the drag material is malleable. The drag material may also beeliminated. The line may be of various lengths and may be made ofvarious materials to be more or less abrasive and more or lessabsorptive. In a particular embodiment, the line comprises twocomponents, a string and a sampling cloth. The string component may bereferred to as the proximal segment, whereas the sampling cloth may bereferred to as the distal segment. The string may be made of, forexample, an absorbent fiber, mesh, or a textured fiber. In oneembodiment, the string and sampling cloth are constructed of the samematerial. The line and the sampling cloth may be of various proportions.In some embodiments, a portion of the string may be pulled out of thecapsule prior to the subject swallowing the capsule, and the end of thestring may be attached to the cheek. After the predetermined period oftime, the string may be removed from the esophagus.

U.S. Pat. No. 6,475,145, incorporated by reference, discloses aretrievable and ingestible radioactive capsule swallowed by a subjectand held in place by means of a cord for diagnosis of GERD andacid-reflux. The radioactive capsule is degraded below pH4 so themeasure is a radioactive release. The cord provides a means forretrieving the capsule. U.S. Pat. No. 5,738,110, incorporated byreference, describes a device for the diagnosis of certaingastrointestinal pathogens. This device comprises a gelatin capsulewhich contains a drag material which has thin sample cloth embeddedtherein, which is attached to a string. The patient holds the string andswallows the capsule, allowing the sample cloth to come into contactwith the stomach. Both of these devices may be adapted for use in thepresent invention.

In another embodiment, the device further comprises a capture agent forone or more diagnostic indicators. These capture agents may be locatedon the line. In a particular embodiment, the capture agents may belocalized on the proximal segment of the line. The term “capture agent”refers to an agent that binds an analyte through an interaction that issufficient to permit the agent to bind and concentrate the analyte froma homogeneous mixture of different analytes. The binding interaction maybe mediated by an affinity region of the capture agent. Representativecapture agents include antibodies, and more specifically monoclonalantibodies. Further non-limiting examples include eosinophil granuleprotein antibodies. The phrase “surface-bound capture agent” refers toan agent that is immobilized on a surface of a solid substrate, wherethe substrate can have a variety of configurations, e.g., a string.

The predetermined period of time may be various lengths of time. Forexample, the predetermined time may be between 15 minutes and 12 hours.In some embodiments, the predetermined period of time may be 1, 2, 3, 4,5, 6, 7, 8, 9, 10, 11, 12, 18, 24 or more hours, or any incrementthereof. In one embodiment, the predetermined period of time is 15minutes. In another embodiment, the predetermined period of time is 1hour. In a further particular embodiment, the predetermined period oftime is 12 hours.

a. The Esophageal String Test (EST)

The Enterotest™ (FIG. 2) (HDC Corporation) was developed as a minimallyinvasive method to assess for small intestinal parasites, e.g., Giardialamblia (Gracey et al., 1977; Thomas et al., 1974). After the capsule isswallowed, the distal end of the nylon fiber string (nearest the gelatincapsule) (FIG. 2A-bottom arrow) resides in the lumen of the smallintestine, and adherent mucous, secretions and parasites (if present),adhere to the nylon string and upon stripping from the string, can bedetected by light microscopy. The anatomical location of the string canbe identified by the provided pH indicator that is touched to portionsof the string to identify where boundaries between acid and alkalinesections of string and thus where the esophageal, gastric and smallintestinal Enterotest sections resided (FIG. 2B). In the rare patientswho are achlorhydric or who are on acid blockading medications, thestandardized formula would be used in the placement of pH monitors tocalculate esophageal length (Mekjavic I B, Rempel et al., 1990).

It is expected that the more proximal end of the string residing withinthe esophagus will capture inflammatory markers from esophagealsecretions, as well as squamous epithelial and inflammatory cells, i.e.eosinophils, present in microabscesses (FIG. 6), thus allowing for aminimally invasive measurement of esophageal inflammatory disease andremission in EE. In the Esophageal String Test (EST), the thinnerproximal end of the Enterotest string™ (FIG. 2—top arrow) is taped tothe patient's cheek, and the capsule is swallowed, leaving a trail ofstring in its path through the esophagus and into the duodenum (FIG.2—bottom arrow). Within a short period of time, the gelatin capsuledissolves and the end of the string is left free in the duodenum. TheEST is left in place for between 15 minutes and 12 hours and is thenremoved for analysis as described below. Herein, evidence that the ESTprovides a sensitive and specific method for analyzing esophagealluminal markers of inflammation at the protein and mRNA levels ispresented. The original Enterotest™ will be used without anymodifications.

Endoscopic and histopathological representations relevant to this testare shown in FIG. 6. A significant percentage of patients with EE showevidence of superficial eosinophil-rich exudates as evidenced by thegross endoscopic view (FIG. 6A) (Sundaram et al., 2004) andcorresponding histological section (FIG. 6B). Even if there is no grossevidence of exudation, intact and degranulating eosinophils andeosinophil microabscesses are routinely present along the superficialepithelial surface allowing for assessment of their secreted mediators(FIG. 6C) by the EST (Straumann et al., 2004; Walsh et al., 1999).Importantly, the 4-6 mucosal biopsies usually obtained for EE diagnosiscapture only a small fraction ((<0.01%) of the total surface area of theesophagus and may be highly variable in their findings. For example,histological sections from 3 biopsies of the same patient taken atdifferent locations during the same endoscopy show: (1) normal appearingepithelium (FIG. 6D), (2) epithelium with minimal eosinophilicinflammation (<1 eosinophil/HPF) (FIG. 6E) and (3) a more distal sitewith the typical diagnostic appearance of EE with large numbers ofeosinophils (>15/HPF) (FIG. 6F). This discontinuity in eosinophilicinflammation can be easily missed by biopsy, thus making the proposedEST, which would provide a more holistic measure of inflammation throughthe entire length of the esophageal lumen, a more complete and valuableassessment.

2. Analyzing the Device

Following removal of the device from the esophagus, the device isanalyzed for a diagnostic indicator. As explained above, the line may bemade of various materials to be more or less abrasive and more or lessabsorptive. In some embodiments, the indicator is adsorbed in thedevice. In another embodiment, the indicator binds to the outside of thedevice. In such embodiments, the collected indicators may be scraped offof the sampling cloth for analysis. In yet a further embodiment,secretions may adhere to the device. Secretions and cells can beexamined by a number of different techniques known in the art. Thepresence of inflammatory proteins, RNA or cells can be analyzed withinthe contents removed from the string. As noted above, in one embodimenta capture agent for one or more diagnostic indicators may be present onthe device, wherein the diagnostic indicator may bind to the captureagent. In some embodiments, the device is analyzed for the presence ofone or more diagnostic indicators. In other embodiments, the device isanalyzed for the level of one or more diagnostic indicators. Analysismay be performed by a number of methods, including but not limited toELISA, cytology, mass spectrometry, gas chromatography, Western Blot,Mesoscale, Licor, RNA and DNA extraction, immunohistochemical analysis,and microbial culture and staining. Some of these methods, such asmesoscale and Licor, are recently described technologies and aredescribed in, for example, Savidge et al. (2007) and Gowan et al.(2007). The other methods for analyzing the products are well known inthe art, and are described in, for example, Sambrook et al. (2001) andAusubel et al. (1994).

3. Diagnostic Indicators

The diagnostic indicator may be any factor that indicates the presenceor severity of inflammation of the esophagus. The diagnostic indicatormay be an eosinophil granule protein, a cytokine or chemokine, acellular infiltrate, pH, or a marker of an allergic response. Otherdiagnostic indicators include peripheral and plasma eosinophil counts,mast cells, including leukotrienes. In other aspects, diagnosticindicator comprises one, two or more markers.

a. Eosinophil Granule Protein

Eosinophil granule proteins include major basic protein (MBP),eosinophil cationic protein (ECP), eosinophil peroxidase (EPO), andeosinophil derived neurotoxin (EDN). These proteins are secreted oneosinophil stimulation and actively participate in the subsequentinflammatory response. Eosinophil granule proteins also include otherbiologically active products including bacterial permeablizing protein(BPI) and anti-microbial proteins. Secretion and extracellulardeposition of eosinophil granule proteins in tissues affected byinflammatory diseases suggests that MBP, ECP, EPO, and EDN mayparticipate in the pathogenesis of the inflammatory process. Furtherevidence has demonstrated that the topical application of MBP isassociated with tracheal smooth muscle contraction and ion secretion. Ithas been demonstrated that one of these proteins, in particular, MBP canstimulate the release of the inflammatory cytokine IL-8 from stromalcells within the gastrointestinal tract. In addition, the deposition ofmajor basic protein has been found in inflamed tissues of patientsaffected with colitis and esophagitis.

b. Cytokines and Chemokines

Clinical relevance for many cytokines and chemokines have now beenestablished. Cytokines include the interleukins (IL-1α, IL-1β, IL-2,IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-11, IL-12, IL-13,IL-14, IL-15, IL-16, IL-17, IL-18, IL-19, IL-20, IL-21, IL-22, IL-23,IL-24, IL-25, IL-26, IL-32) and interferons. In particular, a diagnosticfactor may be a Th2 cytokine (e.g., IL-4, IL-5, IL-13). Chemokinesinclude, for example, the eosinophil-specific chemokines eotaxin-1, -2,-3.

c. Marker of an Allergic Response

A marker of an allergic response may also indicate the inflammation ofand disease of the esophagus. An “allergic response” as used herein is adisorder in which the host's immune response to a particular antigen isunnecessary or disproportionate, resulting in pathology. Allergiesarising from an allergic response include, but are not limited to,allergies to pollen, ragweed, shellfish, any food product, domesticanimals, (e.g., cats and dogs), B-venom, and the like. A subset ofallergic responses produce asthma. Allergic asthmatic responses are alsoincluded within the definition of the term “allergic response.”

A marker of an allergic response may be, for example, immunoglobulin E(IgE), tryptase, receptors (FcRI, CD23) or an allergen. CD23 is alow-affinity receptor for IgE, expression of which is induced bycytokines associated with allergic responses. Other inflammatory markersthat may be examined may include, for example, arachadonic acid productsand neurotransmitters such as substance P and bradykinin.

4. Evaluation of Diagnostic Indicators

The diagnostic markers are evaluated to diagnose a disease of theesophagus. Evaluation may include the presence or absence of anindicator that allows for diagnosis of a disease. For example, if asample shows evidence of an inflammatory reaction, this may indicate thepresence of eosinophilic esophagitis. Alternatively, the level of anindicator may be evaluated to either diagnosis or evaluate the level ofseverity of a disease. For instance, the evidence may be increasedlevels of eosinophil granule proteins that may indicate the presence ofEE. In other non-limiting embodiments, increases in specific cytokines,such as IL-6, indicates the presence of GERD. The diagnostic markers mayalso be evaluated to assess the treatment of a disease.

B. Diseases of the Esophagus

The present invention may be used to diagnose or assess treatment of adisease of the esophagus. The disease of the esophagus may be anydisease that comprises inflammation of the esophagus.

1. Gastroesophageal Reflux

In certain aspects, the disease may be gastroesophageal reflux (GERD).GERD is defined as chronic symptoms or mucosal damage produced by theabnormal reflux in the esophagus. Symptoms may include heartburn,inflammation in the esophageal lining, strictures, dysphagia and chronicchest pain. Methods of diagnosis include barium swallow X-rays,esophageal manometry, 24-hour esophageal pH monitoring, andesophagogastroduodenoscapy. Treatments include food and lifestylemodifications, positional therapy, drug treatment and surgery. Thecurrent invention may be used to assess and diagnose GERD orcomplications associated with GERD such as Barrett's esophagus orcancer.

2. Eosinophilic Esophagitis

In another embodiment, the disease may be Eosinophilic esophagitis (EE).EE, an increasingly recognized eosinophilic gastrointestinal disease(EGID), accounts for ˜50% of dysphagia and food impaction. It has beenproposed that food allergy is the underlying etiology of EE. Recenttranslational studies show that skin prick tests (SPTs) are currentlythe best available tool to identify the triggering food allergensand >90% of patients respond to dietary interventions, thus supporting arole for humoral (IgE) and/or cell-mediated food allergy. In addition,esophageal mast cells are significantly increased in EE compared tonormal controls and gastroesophageal reflux disease (GERD). Notably, ofvarious dysregulated genes identified by DNA microarray studies, fivemast cell genes were highly induced, including the high-affinity IgEreceptor (FcεRI) and mast cell tryptase-α. However, the role ofIgE-mediated mast cell (or basophil) responses in the esophagealinflammatory cascade in EE remains uncertain, as noted by the fact thatRAST testing alone shows poor specificity for identifying offendingfoods. To date, no studies have prospectively examined the role of IgEin the esophageal microenvironment associated with EE.

EE is a disorder of the esophagus characterized by esophageal and/orupper gastrointestinal tract symptoms in association with esophagealmucosal biopsy specimens containing high amount of intraepithelialeosinophils within the esophageal squamous epithelium or deeper tissuelevels and normal pH monitoring. EE affects males more than females, andthe diagnosis is typically made in adults during the third and fourthdecades of life, although it may be diagnosed at a later age. Inchildren, the diagnosis is made after infancy and through adolescencewith no recognized peak age of onset. Symptoms may include chest pain,heartburn, dysphagia, food impaction and a lack of responsiveness toacid reducing medications. Treatment of EE involves eithercorticosteroids or elemental diet and not surgery.

Standard of care for EE patients includes initial esophageal endoscopywith biopsy to determine the numbers of epithelial eosinophils (≧5/hpfbeing diagnostic). Since consequences of chronic eosinophilicinflammation in EE can include esophageal remodeling with subsequentesophageal narrowing, trachealization and strictures, therapeuticefforts are typically devoted toward inducing clinical as well ashistological remission. While overall relatively safe, esophagealendoscopy entails procedural risks, is expensive, time consuming and islimited to procuring a 3 mm sample.

To date, no serological, stool or non-invasive tests have provideddurable results correlating histological evidence of disease progressionor remission in EE. Presently, the state of esophageal inflammation inpatients with EE can only be assessed with an invasive endoscopy.Although the cost-benefit ratio is unknown, repeated endoscopies withbiopsies are the best test tool date to assess disease status andresponse to treatment.

To address this issue, an Esophageal String Test or EST, such as theEnterotest™ a string-based test first used for detection of Giardiainfections, can be used in its native form to assess esophagealinflammation at both the protein and mRNA levels and may potentially beused to monitor disease activity. The EST may offer a minimally invasivemethod to assess the presence of inflammation associated with activedisease.

a. Eosinophilic Esophagitis and Food Allergies

Food allergic diseases affect between 4-6% of children in the UnitedStates per year. During the last decade, an increasing number ofchildren developed a new manifestation of food allergy termedeosinophilic esophagitis (EE) (Furuta et al., 2007). Several lines ofevidence support a close relationship of EE with food allergic diseases.First, skin prick testing (SPT), a reliable indicator of IgE-mediatedfood reactions, correlates consistently with esophageal inflammation inEE (Spergel, 2007). Second, patients with EE often associate symptomsfollowing the ingestion of specific foods. Furthermore, specificelimination of those foods and/or foods identified by SPTs leads toclinicopathological remission in EE (Spergel et al., 2002; Kagalwalla etal., 2006). Third, mucosal biopsies from patients with EE demonstratesignificantly increased numbers of mucosal mast cells compared to thosefrom patients with gastroesophageal reflux disease (GERD) or normalsubjects, suggesting their participation in the pathogenesis of thisdisease (Kirsch et al., 2007). Finally, previous work suggests thatpatients with eosinophilic gastrointestinal diseases (EGIDs) and foodallergy demonstrate increased expression of CD23 on intestinalepithelial cells and in stool samples (Li et al., 2006). While theprecise role of CD23 in food allergic responses is not certain, recentstudies suggest that the human CD23a isoform participates as abidirectional transporter of both free IgE and IgE/antigen complexes,and can potentially deliver IgE and its bound allergen across intestinalepithelial cells to induce mast cell activation (Montagnac et al.,2005a; Montagnac et al., 2005b; Bevilacqua et al., 2004; Yu et al.,2003). Taken together, these observations provide strong evidencesupporting a role of food allergic responses (including those mediatedby IgE) in the pathogenesis of EE.

b. Diagnostic Criteria for Eosinophilic Esophagitis

The recent emergence of EE is emphasized by the fact that diagnosticcriteria have only recently been established (Furuta et al., 2007).Clinically, EE is characterized by symptoms including abdominal pain,regurgitation, feeding intolerance, food impaction and dysphagia.Histologically, esophageal biopsies contain large numbers ofintraepithelial eosinophils (≧15 eosinophils/high power field), oftenwith eosinophil microabscesses and luminal layering. These findings areunresponsive to acid blockade, e.g. proton pump inhibition, but dorespond to elimination (or elemental) diets and corticosteroids. Thisdisease does not affect the columnar epithelium of the stomach or smallintestine. Thus, when a patient has persistent symptoms that areassociated with esophageal epithelial eosinophilia and normal gastricand duodenal mucosa, and gastroesophageal reflux (GERD) and other causesof eosinophilia have been ruled out, the diagnosis of EE can be madewith confidence. The importance of clear diagnostic criteria isemphasized by the fact that many patients are now receiving thediagnosis of EE based on histological findings alone, without properinvestigations to exclude other causes of esophageal eosinophilia (Gentaet al., 2007; Ngo et al., 2006).

c. Pathogenesis of Eosinophilic Esophagitis

Although a number of factors relate food allergic responses to EE, theexact mechanisms defining the pathogenesis of EE remain uncertain (Hoganand Rothenberg, 2006). Current paradigms of potential mechanismsunderlying EE address several different pathways including, the role ofIgE in EE (Foroughi and Prussin, 2005), identifying the mechanisms ofsquamous eosinophilia (Blanchard et al., 2006a), and defining whetherthe associated phenotype of patients is predominantly Th1 or Th2(Straumann et al., 2001; Straumann et al., 2005). A few translationalstudies have addressed the participation of IgE-mediated immediatehypersensitivity responses in EE. Increased numbers of mast cells(Kirsch et al., 2007), CD23 expression on intestinal epithelial cells(Li et al., 2006), and historical evidence of other IgE-mediatedallergic diseases (Spergel, 2005), provide circumstantial evidencesupporting a role for IgE-mediated responses in EE. Alternatively, notall patients have elevated total or specific IgE, show evidence ofatopic disease, or have increased mast cells in their esophageal tissue(Furuta et al., 2007). Typically, patients do not complain of immediatereactions associated with the ingestion of candidate foods, and do notpresent with systemic allergic symptoms involving other organs such asthe lung or skin, although EE has been referred to by some as “asthma oreczema of the esophagus” (Arora and Yamazaki, 2004).

A recent translational study focused on one potential mechanism thatdrives eosinophils into the esophageal squamous epithelium (Blanchard etal., 2006a). A DNA microarray study showed that the eosinophil-specificchemoattractant eotaxin-3 (CCL26) was the most highly up-regulated genein the squamous epithelium from biopsies of patients with EE compared tothose with GERD and those with normal mucosa. In this report, studiesusing eotaxin receptor CCR3-deficient (knockout) mice confirmed thatesophageal eosinophilia, in an IgE-dependent intranasal allergen model,was dependent on the presence of CCR3 for eosinophil recruitment to theesophagus. Finally, 32.1% of EE patients compared to 22.4% of non-EEmatched subjects in this study had a single nucleotide polymorphism(SNP)+2,496 T→G in the eotaxin-3 gene that was associated with diseasesusceptibility. Thus, eotaxin-3 remains the only biomarker to dateassociated with EE. However, since only a limited percentage of patientspossessed this eotaxin-3 SNP, it is likely that other relevantbiomarkers will be identified.

Basic and clinical studies suggest that the preponderance ofinflammatory responses associated with EE is of a Th2 phenotype. Asdemonstrated in murine models utilizing IL-5 and eotaxin-1 null mice,esophageal eosinophilia is dependent on IL-5 expression and partiallydependent on eotaxin-1 (Mishra et al., 2001; Mishra et al., 2002).Histological staining of affected esophageal epithelium shows increasedIL-5 staining (Straumann et al., 2001; Straumann et al., 2005) and arecent small clinical series showed that anti-IL-5 antibody leads to theresolution of clinical and histological findings in some EE patients(Stein et al., 2006). While food allergen-induced responses leading toover-expression of eotaxin-3 by the squamous epithelium in the esophagusis likely involved in the Th2 inflammatory cascade, the precisepathogenesis of esophageal eosinophilia in EE has yet to be established.To date, eotaxin-3 is the only potential biomarker that has beenidentified for EE.

d. Problems Associated with Care of Patients with EE

While treatments of EE have become well accepted in terms of the use ofnutritional elimination diets (Kelly et al., 1995; Markowitz et al.,2003; Spergel et al., 2005), including elemental diets, andcorticosteroid administration (Faubion et al., 1998; Liacouras et al.,1998), the precise treatment endpoints are much less clear (Furuta etal., 2007). For instance, some clinicians treat to resolve onlysymptoms, while others treat to induce both clinical and histologicalremission, i.e. treat to decrease numbers of esophageal eosinophils(Aceves et al., 2008). This decision is often shaped by the reluctanceto have patients undergo repeated esophagogastroduodenoscopy (endoscopy)and biopsies, and by uncertainty about the natural history and incidenceof long-term sequelae of chronic esophageal eosinophilia that includeesophageal remodeling, subepithelial fibrosis and narrowing. Those whofeel that chronic esophageal eosinophilia leads to esophageal fibrosisand stricture formation treat patients and perform repeat endoscopy withbiopsy to insure that mucosal eosinophilia has resolved. Those who feelthat chronic esophageal eosinophilia will not lead to esophagealstrictures will treat with symptom relief as the only endpoint and notrepeat endoscopy with biopsy. Thus, a significant number of EE patientswho never undergo post-treatment sampling of their mucosa may continueto have mucosal disease that could possibly leave them vulnerable to thelong-term complications of chronic esophageal eosinophilia.Alternatively, patients may not develop complications, but whichpatients will or will not develop these complications, how long it mighttake, and where the complications will develop anatomically are entirelyunknown, since the natural history of EE has not been adequatelycharacterized. Thus, the cost-benefit analysis of repeated endoscopieshas not been determined.

In this regard, the obvious problem is that the only method currentlyavailable to determine the state of esophageal mucosal inflammation isendoscopy with mucosal biopsy (Gonsalves et al., 2006). While thisprocedure is relatively safe overall, several downsides do existincluding the potential complications of conscious sedation or generalanesthesia (hypoxia, allergic reactions to the medications, airwaycompromise) and risks of the procedure itself (esophageal perforation,bleeding and infection). Endoscopy with biopsy is limited in that eachbiopsy only provides an assessment of <0.001% of the total esophagealsurface area. Endoscopy is costly and often is not covered by insurancecompanies when repeated as described above. It is also important torecognize that while EE is currently a recognized disease amongst mostclinicians, an ICD-9 code for EE does not yet exist. This lack of“certification” of EE has created administrative confusion in terms ofhow to pay for endosocopy. Finally, endoscopy results in lost time fromschool or work. Other proposed methods to analyze the esophageal mucosainclude monitoring symptoms, radiological studies, and serum or stoolanalyses (Gupta et al., 2005). It is a well-recognized fact thatsymptoms do not necessarily correlate with evidence of histologicalactivity (Liacouras et al., 2005). To date, no serological or stoolanalysis has provided reliable and durable findings that correlate withand are consistently predictive of histological evidence of diseaseremission or progression. Aside from a recent study showing correlationsfor measurements of absolute eosinophil counts, plasma EDN and eotaxin-3levels with disease status (Konikoff et al., 2006), preliminary studiesmeasuring eosinophil granule cationic proteins in the serum of affectedpatients have failed to find significant correlations with diseaseactivity. Taken together, these findings indicate that a minimallyinvasive, inexpensive, safe, reliable and accurate method for directmeasurement of esophageal inflammation is needed for initial diagnosisand post-treatment management of patients with EE.

e. Novel Minimally Invasive Method for Assessment of EsophagealInflammation in Patients with EE

The Enterotest™ was first used as a minimally invasive method toidentify infectious organisms (Giardia lamblia) in the duodenum ofsuspected patients (Gracey et al., 1977; Thomas et al., 1974). TheEnterotest™ consisted of a weighted gelatin capsule that was filled witha 90 cm nylon string. The end of the string extruded out of one end ofthe capsule (see FIG. 1). The patient was asked to swallow the capsulewhile holding on to the end of the extruded string. As the capsulepassed through the esophagus, stomach and duodenum, it left a trail ofthe string, the end of which was taped to the patient's cheek. After aperiod of time, the gelatin capsule dissolved, leaving the string freein the duodenum to which parasites within the intestinal mucosa wouldadhere. At a defined period of time, the string was then pulled back outof the mouth and the mucous and secretions on the distal end of thestring were assayed for the presence of adherent parasites.

The proximal part of the Enterotest™ string could be used to assessesophageal inflammation associated with EE and have referred to this asthe Esophageal String Test (EST) throughout this proposal. Benefits ofthe EST include: (1) ease of administration—no preparation/anesthesiarequired, (2) inexpensive—approximately <$20/capsule, (3)expansive—allows for evaluation of the entire length of the esophagus,not just a few sampling sites (4 biopsies=<0.01% of esophageal surfacearea (4) safe—the only side effect may be discomfort or gagging when thecapsule is first swallowed, (5) easily accessible—does not require anendoscopy suite. Preliminary results show that: (1) blood eosinophilsand their granule cationic proteins interact with the EST in vitro andcan be measured in a dose-response manner, (2) further in this regard,the EST detects increased secretion of granule cationic proteins aselaborated by IL-5-activated compared to resting blood eosinophils, and(3) in another esophageal inflammatory disease (GERD), both inflammatoryproteins and mRNA (i.e., IL-8) can be measured in vivo using the EST,and correlates with the degree of esophageal inflammation.

C. DEFINITIONS

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood by those of ordinary skillin the art to which this invention pertains.

The term “assessing” includes any form of measurement, and includesdetermining if an element is present or not. The terms “determining,”“measuring,” “evaluating,” “assessing” and “assaying” are usedinterchangeably and may include quantitative and/or qualitativedeterminations.

The phrase “diagnosing or monitoring a disease of the esophagus” as usedherein refers to a method or process of determining if a subject has ordoes not have a disease of the esophagus, or determining the severity ordegree of a disease of the esophagus.

The term “subject” as used herein refers to any member of the animalkingdom, preferably a human being.

The terms “treatment,” “treating,” “treat,” and the like, refer toobtaining a desired pharmacologic and/or physiologic effect. The effectmay be prophylactic in terms of completely or partially preventing adisease or symptom thereof and/or may be therapeutic in terms of apartial or complete cure for a disease and/or adverse affectattributable to the disease. “Treatment,” as used herein, covers anytreatment of a disease in a mammal, particularly in a human, andincludes: (a) preventing the disease from occurring in a subject whichmay be predisposed to the disease but has not yet been diagnosed ashaving it; (b) inhibiting the disease, i.e., arresting its development;and (c) relieving the disease, i.e., causing regression of the diseaseand/or relieving one or more disease symptoms. “Treatment” is also meantto encompass delivery of an agent in order to provide for apharmacologic effect, even in the absence of a disease or condition.

Various biochemical and molecular biology methods referred to herein arewell known in the art, and are described in, for example, Sambrook etal. (2001) and Ausubel et al. (1994).

D. EXAMPLES

The following examples are included to demonstrate particularembodiments of the invention. It should be appreciated by those of skillin the art that the techniques disclosed in the examples which followrepresent techniques discovered by the inventor to function well in thepractice of the invention, and thus can be considered to constitutepreferred modes for its practice. However, those of skill in the artshould, in light of the present disclosure, appreciate that many changescan be made in the specific embodiments which are disclosed and stillobtain a like or similar result without departing from the spirit andscope of the invention.

Example 1

The string test was administered to a patient with reflux esophagitisand left in place for 1 and 12 hours. The first 10-20 cm of the stringwas pulled out from the capsule and the end of the string was securedbetween the fingers. The capsule and remaining string were swallowedwith water and the string in the fingers was taped to the cheek. Afterthe predetermined period, the taped string was removed from the cheekand the string retrieved from the intestinal tract. Following removal,the contents of the string were assessed for the presence ofinflammatory proteins and RNA. The entire string was placed either inprotein buffer, RNA stalizing solution or formalin. After 1 and 12 hoursin the esophagus, elevations of interleukin-8 and TNF α-protein wereidentified by Mesoscale and ELISA technology. In these studies, proteinwas extracted from the string, and the soluble product was placed in thewell of a 96-well plate for analysis. Following placement of the solubleproduct in the well of the plate, a series of incubations and washeswith target antibodies and buffers took place. Finally, the plate wasplaced in a specialized detection equipment for quantitation.

Example 2

Identification of microbial flora from EST. Identification andenumeration of esophageal and oral bacterial population by selectiveculture.

Microbial culture. Strings were swallowed or remained in the mouth for15 min. A 5 cm piece of esophageal or oral string was asepticallytransferred to 5 ml of sterile Hank's Buffered Salt Solution (HBSS) andvortexed for 30 sec before a 5 min incubation at RT. For nasal secretioncollection swap was humidified in sterile saline; a nasal swab wascollected using a BBL™ culture swab. String and nasal solution wasvortexed again for 30 sec and the string removed. The solution wascentrifuged for 15 min at 4750 rpm. Supernatant was aspirated and thepellet was resuspended in 1 ml of sterile HBSS. Dilutions up to 10⁻⁵were performed and 50 ml of non diluted samples or 10⁻⁵ dilution wasplated in duplicate onto selective media. Plates were incubated at 37°C. overnight in aerobic or anaerobic (BD Bioscience Gas Packs)conditions. Plate colonies were then enumerated and total Colony FormingUnits (CFU) were calculated. Staphylococcus sp, Streptococcus sp,Lactobacillus and Peptostreptococcus were cultured respectively onMannitol salt agar, Selective Streptococcus agar, Difco lactobacilli MRSagar and CDC anaerobe blood agar with Phenyl Alcohol (Becton Dickinson).See FIG. 3.

DNA purification. Strings were incubated in 180 ml of ATL lysis buffer(QIAGEN) containing 2 mg/ml lysozyme (Sigma), bacteria were lysed for 1hour at 37° C. Proteinase K was added in 200 ml of AL lysis buffer(QIAGEN) and incubated for 1 hour at 56° C. Purification of total DNAwas performed following the manufacturer's instructions (DNeasy Bloodand Tissue KIT QIAGEN). See FIGS. 4A-B.

Example 3

Measurements of eosinophil derived granule proteins from the EsophagealString Test (EST) can detect histological evidence of esophagealinflammation associated with EE. Preliminary results show thatsupernatants derived from activated eosinophils adhere to the EST andthat eosinophil derived granule proteins within these samples can bemeasured by Western blot and ELISA analyses. These findings support thetechnical feasibility for the EST to be used to measure eosinophilderived granule proteins contained in the esophageal lumen of patientswith EE.

Detection of eosinophil granule major basic protein-1 (MBP1) on ESTstrings incubated with acidic lysates of resting eosinophils. In orderto demonstrate the ability of EST string to detect eosinophil granulecationic proteins, purified blood eosinophils from normal subjects (>99%eosinophils) were used to prepare soluble acidic lysates by sonicationof different numbers of eosinophils (1×10⁵ or 1×10⁶ cells) in either0.1N HCl (pH 3) routinely used to solubilize eosinophil granule cationicproteins from secondary granules, and a 25 mM sodium acetate/acetic acid(NaOAc) buffer (pH 4.3) routinely used for size exclusionchromatographic purification of these proteins. EST nylon fiber strings(2 cm lengths) were incubated with 0.5 ml of the eosinophil sonicatesfor 1 hr, the strings removed and blotted briefly until visibly dry,boiled in SDS-PAGE sample buffer, and analyzed by SDS-PAGE/Westernblotting for the presence of MBP1 adsorbed to the strings (FIG. 7). Ofnote, the strings were purposefully not washed prior to boiling inSDS-PAGE sample buffer so as to simulate in vivo diagnostic performanceof the Enterotest™ EST, in which strings are pulled from the GI tractand directly analyzed without further washing for the presence ofadherent mucus and Giardia lamblia parasites. After 1 hr incubation,MBP1 could be detected on EST strings by Western blotting usingsonicates prepared from 1×10⁵ eosinophils, with increased MBP 1 detectedfor sonicates of 1×10⁶ eosinophils, both HCl and acetate bufferproviding comparable MBP1 levels. These results demonstrate the abilityof EST string to detect eosinophil MBP1 from soluble lysates of as fewas 2×10⁵ eosinophils/ml.

Time course for EST string detection of soluble eosinophil granulecationic proteins. In order to determine the minimum amount of timerequired for detection of soluble eosinophil granule cationic proteinsusing the EST, 2 cm lengths of EST string were incubated with 0.5 ml oflysates of purified blood eosinophils that were prepared by sonicationof 1×10⁶ eosinophils in either 25 mM sodium acetate/acetic acid (NaOAc)buffer (pH 4.3) or in phosphate-buffered saline (PBS; pH 7.3) ESTstrings (2 cm lengths) for 1-16 hours. The strings were harvested byblotting to dryness as above, boiled in SDS-PAGE sample buffer, andanalyzed by SDS-PAGE and Western blotting for the presence of EPO andMBP1 adsorbed to the strings (FIG. 8). The upper two panels from thesame Western blot show both the high (52 kD) and low (14 kd) molecularweight subunits of EPO, and the lower panel shows results for MBP1. BothEPO and MBP1 were detectable on the EST strings within 1 hr, withsomewhat increased amounts of the proteins detected using lysatesprepared in PBS compared to the acidic sodium acetate buffer. Incubationtimes longer than 1 hr did not significantly increase the amount of EPOor MBP1 adsorbed to the strings, suggesting that relatively short-termexposure of the EST strings in vivo may be sufficient for detection ofthe eosinophil granule cationic proteins.

IL-5 activation of blood eosinophils increases eosinophil secretion anddetection of eosinophil granule cationic proteins adsorbed to ESTstrings. In order to determine whether the EST can differentially detectand measure granule cationic protein secretion from resting vs.cytokine-activated eosinophils, intact purified blood eosinophils (1×10⁶cells) were incubated with 2 cm lengths of EST string for 1 hr in 0.5 mlof complete tissue culture media containing 8% FBS with or without theaddition of IL-5 (25 ng/ml) to activate eosinophil secretion of MBP1 andEPO. The strings were harvested from the TC media, blotted to removeexcess media as in the above experiments. Again, the strings were notwashed to simulate their use in vivo, but half the strings were brieflyincubated in trypsin/EDTA solution to remove adherent eosinophils.Strings were then boiled in SDS-PAGE sample buffer and analyzed bySDS-PAGE/Western blotting for EPO and MBP1 (FIG. 9). Both MBP1 and thehigh (˜52 kD) and low (14 kD) molecular weight subunits of EPO weredetectable by Western blotting (FIG. 9, left). Of note, activation ofthe eosinophils with IL-5 modestly increased the amount of both MBP 1and EPO detected on the EST strings (left and as quantitated on theright). Some of the MBP1 and EPO signal was due to IL-5 activatedeosinophils (visualized by inverted phase-contrast light microscopy)that were adherent to and intercollated between the nylon fibers of thestrings—data not shown), as evidenced by the decreased MBP1 and EPOsignals for strings treated with trypsin/EDTA to remove the adherentcells. Importantly, these findings demonstrate the utility of the ESTfor detection of eosinophils and their secretion of granule cationicproteins as induced by eosinophil-active cytokines such as IL-5.

Time course for EST detection of IL-5-induced granule protein secretionby blood eosinophils. In order to determine the minimum amount of timerequired for EST detection of IL-5-induced eosinophil granule proteinsecretion, blood eosinophils (1×10⁶ cells @>99% purity) from normalsubjects were activated with IL-5 (25 ng/ml) in the presence of ESTstrings (2.0 cm/time point) for 1-16 hours in 0.5 ml RPMI1640 tissueculture media containing 8% FBS. As above, strings were blotted todryness and transferred to SDS-PAGE sample buffer, boiled and analyzedby SDS-PAGE/Western blotting using antibodies to EPO, MBP1 andCLC/Gal-10 (FIG. 10). These studies included Western blotting forCLC/Gal-10, since this highly expressed eosinophil cytosolic and primarygranule protein constituent comprises an estimated 7-10% of totaleosinophil protein (Ackerman et al., 2002; Ackerman et al., 1994;Ackerman et al., 1993), is the second most abundantly expressed mRNA indeveloping eosinophils (Plager et al., 1999), and most importantly, wasrecently shown to be amongst the 20 most highly up-regulated genes incDNA microarray analyses of esophageal biopsy tissues obtained frompatients with EE compared to GERD and normal control subjects (Blanchardet al., 2006b). Results showed that CLC/gal10, along with MBP1 and EPO,were detected by EST within 1 hour of incubation of the strings withIL-5 activated eosinophils. For reasons that are unclear, the amounts ofall three proteins detected decreased somewhat after 2-4 hours ofeosinophil-string incubation, but by 16 hrs returned to levelscomparable to those obtained after 1 hour. These results importantlydemonstrate the ability of the EST approach to detect IL-5-inducedsecretion of three key eosinophil biomarkers in vitro, two of the majorgranule cationic proteins, and cytosolic CLC/Gal-10, recently shown tobe markedly increased at the mRNA level in eosinophilic esophagitis(Blanchard et al., 2006b; Blanchard et al., 2006c).

The EST can be used to detect IL-5-induced eosinophil granule proteinsecretion by ELISA assay. In order to determine whether the EST can bepaired with more sensitive, quantitative and high throughput assays foreosinophil-associated biomarkers, e.g. ELISA assays, the above ESTexperiment was repeated and IL-5-induced eosinophil secretion ofeosinophil-derived neurotoxin (EDN) was measured by ELISA. Bloodeosinophils (1×10⁴-1×10⁶ cells @>99% purity) from a normal subject werecultured ±IL-5 @ 25 ng/ml in the presence of EST string (2.0 cm/0.5 mlcultures) for 1 hour in RPMI 1640 tissue culture media supplemented with8% FBS. To elute and solubilize adherent eosinophils and eosinophilgranule proteins, the strings were removed, blotted to dryness, andtransferred to 0.5% NP-40 elution buffer (pH 6.0) for 30 minutes, withvortexing at room temperature. The IL-5-inducible eosinophil expressionof EDN eluted from the EST strings was analyzed by an EDN-specific ELISA(MBL Co., Ltd) (FIG. 11). As measured by ELISA, IL-5 induced >4-foldmore detectable EDN than eosinophils incubated with EST strings in theabsence IL-5. However, significantly less EDN was measured using 10-foldfewer eosinophils (1×10⁵ cells/0.5 ml/2 cm string), suggestingsensitivity lies between 1×10⁵-1×10⁶ eosinophils under these conditions.Of note, a recent study by Konikoff and colleagues (Konikoff et al.,2006) showed that plasma EDN levels, along with absolute eosinophilcounts and eotaxin-3 levels, were significantly correlated withesophageal biopsy eosinophil density, and may have value as non-invasivebiomarkers for monitoring EE. Overall, these results demonstrate thefeasibility of using more quantitative assays than Western blotting toquantitatively measure eosinophil biomarkers such as EDN on EST strings,supporting the use of ELISAs and more high throughput approaches, i.e.Mesoscale (see FIGS. 12-16), to quantitate Th1 (IL-2, TNF-α, INF-γ) andTh2 (IL-4, IL-5, and IL-13) cytokines and multiple eosinophil-associatedbiomarkers of interest including the eosinophil granule proteins (MBP,EPO, EDN, ECP), CLC/Gal-10, eosinophil-active cytokines (e.g., IL-5) andchemokines (eotaxin-3), some of which e.g., EDN, eotaxin-3) may haveutility as correlates of esophageal eosinophilic inflammation (Konikoffet al., 2006).

Experimental Design and Methods

1. Determine if Measurements of Eosinophil Derived Granule Proteins fromthe Esophageal String

EST can detect histological evidence of esophageal inflammationassociated with EE. Presently, two large voids exist in the field offood allergic diseases in terms of eosinophilic esophagitis (EE) (Furutaet al., 2006). First, the pathogenesis of EE is not certain andknowledge is limited by the fact that repeated endoscopy with biopsiesare necessary for any assessment of the inflammatory state in vivo.While at least one biomarker (epithelial-expressed eotaxin-3) (Blanchardet al., 2006a; Konikoff et al., 2006) has thus far been identified forEE, the EST offers an excellent opportunity to identify novel biomarkersthat will expand the understanding of the pathogenesis of EE in arelatively simple, minimally invasive fashion. Second, clinicalassessment of patients with EE is severely limited, again by the factthat, short of symptomatic assessment, endoscopy with biopsies is theonly method currently available to determine diagnoses and theeffectiveness of treatment. Again, the EST offers a minimally invasivetool for assessing esophageal inflammation. The EST or some modificationthereof may offer diagnostic capabilities, e.g., in the identificationof an EE proteome or transcriptome.

EST addresses these two relevant and timely problems in a novel andunique manner. A goal is to associate the levels eosinophilicinflammation identified in endoscopically obtained mucosal biopsies withthe levels of eosinophil-specific markers, the eosinophil derivedgranule proteins, measured using the EST in children with well-definedEE. EST-detected eosinophil derived granule proteins were used as theprimary marker because of their specificity in the evaluation ofpatients with EE. EST results obtained from patients with EE arecompared with those obtained from patients with normal esophagealhistology to determine the sensitivity of the EST.

Preliminary studies showed that the EST can be used as a novel minimallyinvasive method to analyze esophageal inflammation. The EST bindseosinophils and eosinophil derived granule proteins in vitro in a timeand concentration-dependent fashion that is enhanced by eosinophilactivation with IL-5. In vivo studies demonstrate that the ESTsuccessfully measures protein and mRNA of associated pro-inflammatorycytokines in GERD.

a. Measure Eosinophil-Derived Granule Protein Levels Using ESTs inPatients with EE

EST administration. Any subject in whom an esophageal disease issuspected may be evaluated. Patients will swallow an EST (FIG. 2A) thenight prior to an already scheduled clinically indicated endoscopy (12hour time point). This time point has been established for at leastthree reasons. First, preliminary results demonstrate binding ofeosinophil granule proteins within this time frame in vitro. Second,cytokines associated with inflammatory esophageal disease are able toadhere to the EST following an overnight incubation in situ in theesophagus. Third, since the patient will undergo anesthesia, the patientcan ingest nothing for 4-6 hours before the procedure. While it islikely that the inflammatory markers will adhere to the EST at earliertime points as suggested by the preliminary in vitro and in vivostudies, 12 hours provides a reliable and safe amount of time for theEST incubation. Patients will be asked to swallow a capsule and theattached EST string will be taped to the cheek. At the time of theendoscopy, the doctor or research study nurse will remove the EST.

Processing the EST. Just prior to the endoscopy, ESTs will be removedand cut into pieces according to anatomical location, i.e., mouth,esophagus and stomach. Within the packaging for each EST is a pHindicator, that when applied to the EST, marks the EST with a colorchange that allows for identification of the part of the GI tract thatthe string had resided. For instance, the gastric section of the stringchanges to orange (acidic environment) and the esophageal section isblue green (alkaline environment) (FIG. 2B). The indicator is onlyapplied to a small (<1 cm) part of the string and this portion will notbe included in the analysis. The length of the string from the lips tothe proximal oropharynx to is measured determine the oral section.Sections of the string will be measured to standardize the final data(i.e., pg of protein per ml per cm of string). Sections are then placedin an Eppendorf tube with either sample elution buffer for proteinanalysis or TRIZOL reagent for RNA isolation (Furuta et al., 2005).Samples will be immediately frozen and processed in batches of 10-20 toeliminate day-to-day variability in isolation techniques, and thenstored at −80° C. for final protein or mRNA analysis.

Analysis of the EST. In this experiment, samples derived from the ESTare only analyzed for the presence of eosinophil derived granuleproteins. The reason for this is that it allows a primary set of markersthat are specific for eosinophils. To date, eosinophilic enumeration isthe benchmark for the histological aspect of diagnosis and treatment(Furuta et al., 2007). Measurements and comparisons of otherinflammatory markers (cytokines, leukotrienes, etc.) may offerpathogenetic insights, but their relevance as a specific marker fortissue eosinophils is limited at this point. Thus, only eosinophilderived granule proteins are measured from EST samples and compare themto eosinophilic inflammation identified in the histological specimens.

Protein analyses. The eosinophil derived granule proteins to be assayedin serum (or plasma as appropriate) and in the EST samples include MBP1,EDN, ECP, EPO, and cytosolic CLC/Galectin-10. The eosinophil proteinanalyses will be performed by double antibody RIAs (for MBP1,CLC-Gal-10) as previously performed (Ackerman et al., 1981; Ackerman etal., 1990; Ackerman et al., 1980), commercially available ELISAs (forEDN, ECP), a commercially available luminescence enzyme activity assay(for EPO), and Western blotting (all proteins as needed forconfirmation). For all assays, results will be recorded quantitativelyas protein level/ml of serum or plasma, or protein level/ml/cm stringfor the EST (for ELISAs or RIAs), and blots will be scanned andquantitated by comparisons to purified granule protein standard curves(as needed for Western blotting confirmation). In addition to this panelof eosinophil-specific protein markers, the levels of eotaxin-3 will bedetermined using a commercially available ELISA as previously reportedfor EE patients (Konikoff et al., 2006). Finally, the levels ofeosinophil-expressed pro-fibrotic cytokines and growth factors that maycontribute to the tissue remodeling (epithelial hyperplasia andfibrosis) seen in EE, including IL-1

IL-6 and TGF-

will be measured using commercially available ELISAs as previouslyreported (Gomes et al., 2005) or by the Mesoscale multiplex assaysystem. All assays of the eosinophil granule cationic and other proteinsinclude quality control (QC) samples with known low and high levelconcentrations of these eosinophil proteins to control for inter-assayvariation, and these QC samples will be included in all assays ofpatient plasma and EST samples to standardize the detection limits andvariability of each of the assays.

Statistical Analyses. Prevalence of eosinophil derived granule proteinswill be summarized using the proportion of patients whose EST sampleswith detectable eosinophil derived granule proteins grouped by thepatients with pathologically proven EE and those with normal histology.Ninety-five percent confidence interval will be used to quantify theprecision of these estimates. Assuming that 15% of patients with normalhistology and 60% of EE patients can be successfully enrolled, thesample size will be 150 EE patients and 66 patients with normalhistology. There is no previous data for estimating the prevalence andhence the most variable case (i.e., the prevalence is 50%) is assumedfor precision estimates. For these sample sizes, the error margin wouldbe 12% and 8% respectively for patients with normal histology andpatients with EE.

b. Determine the Associations of Eosinophil Derived Granule ProteinLevels from EST with Histological Inflammation in EE Patients

Assessment of clinical and mucosal inflammation. Clinical symptoms willbe recorded at the time of initial assessment and recorded according tostandardized data sheets that include unidentifiable patient ID number,primary symptom, histopathological data (numbers of eosinophils in eachbiopsy, presence or absence of microabscesses and superficial layering),treatment, and whether primary symptom was affected by treatment (Walshet al., 1999; Desai et al., 2005; Teitelbaum et al., 2002). Results willbe recorded as the presence of primary symptoms and resolution ofsymptoms. Mucosal biopsies will undergo standard blinded analysis bystaff pathologists. Results will be recorded as the number ofeosinophils/HPF and the presence or absence of eosinophil microabscessesor superficial layering. Inter-observer variability will be assessedafter approximately every 50 slides, discrepancies noted and resolved(Walsh et al., 1999; Desai et al., 2005; Teitelbaum et al., 2002; Nurkoet al., 2004).

Statistical Analyses. Chi-square test will be used to assess theassociation of presence of eosinophil derived granule proteins from theEST with the presence of eosinophils/degranulation in biopsy tissue orwith symptoms (yes/no) among EE patients. The discriminating ability ofeosinophil derived granule proteins for the presence of EE or clinicalsymptoms of EE will be summarized with sensitivity and specificity. Theassociation of presence of eosinophils with level of eosinophil derivedgranule proteins (a continuous variable) will also be assessed usinglogistic regression. Receiving operational characteristics (RPC) curvewill be used to examine the discriminating ability of eosinophil derivedgranule proteins for presence of eosinophils and determine the optimumcutoff for clinical use. For the expected sample size, there will be 80%power to detect statistical significance at the 5% level even if the EEpatients have 20% higher prevalence of eosinophil derived granuleproteins than patients with normal histology. Also, there will be 80%power to detect statistical significance if the sensitivity is 15%larger than the usual minimum acceptable cut off of 75% or if thespecificity is 14% higher than the usual minimum acceptable cutoff of80%.

Example 4

Preliminary results demonstrate that the EST can specifically detecthigh levels of IL-8 mRNA and protein associated with reflux esophagitis.The EST can be used as a minimally invasive in vivo test to identifyinflammatory molecules potentially responsible for the pathogenesis ofEE, and to monitor the ability of treatment to modulate thisinflammation.

In vivo assessments of the EST for detection of biomarkers of esophagealinflammation. In order to demonstrate feasibility for using the EST tomeasure protein and mRNA biomarkers of esophageal inflammation in vivo,a number of Enterotest™/EST assessments were performed in a well-definedpatient with a confirmed diagnosis of gastrointestinal inflammation dueto gastroesophageal reflux disease (GERD), demonstrating increasedlevels of IL-8 at both the protein and mRNA levels compared to otherinflammatory cytokines in the proximal and distal esophagus, but not inother locations in the GI tract, and decreased expression of IL-8following treatment with a proton-pump inhibitor. IL-8 has beenpreviously shown to be increased in tissues and BAL fluid associatedwith GERD with levels diminishing following treatment (Isomoto et al.,2004; Oh et al., 2007; Thibeault et al., 2007; Yoshida et al., 2004.These studies demonstrate the ability of the proposed EST toquantitatively measure relevant biomarkers of esophageal inflammationand their decrease during disease remission following treatment,providing proof-of-principle in vivo for its use in the proposedexploratory studies of eosinophilic inflammation and biomarkers ofIgE-mediated responses in patients with EE.

Detection of high levels of IL-8 protein in the esophagus by theEnterotest™ in a patient with GERD. An adult with well-characterizedGastroesophageal Reflux Disease (GERD) was studied. Endoscopic analysiswas grossly normal but mucosal biopsy showed histological evidence ofmild eosinophilic inflammation (FIG. 12A) and basal zone hyperplasia. Inaddition, 24 hour pH monitoring of the distal esophagus was abnormalwith >18% pH<4 and >200 episodes of pH<4 measured, solidifying thediagnosis of GERD. The Enterotest™ was performed and the string removedafter 12 hours and cut into oral, esophageal (proximal and distal),stomach and duodenal segments. These anatomical locations of the ESTsegments were identified by pH measurements on the string (FIG. 2B),i.e., distal section=alkaline pH in the duodenum, middle section=acid pHin the stomach, proximal section=alkaline pH in the esophagus. Proteinsecretions were eluted from the string segments for measurement of IL-8protein using the Mesoscale™ assay (FIG. 12B). Mesoscale is a highthroughput technology that allows multiplex analysis of individualsamples for up to 10 different proteins simultaneously. The resultsclearly show the ability of the EST to detect expression of IL-8 proteinin both the distal and proximal esophagus, but not in the mouth, stomachor duodenum of this patient during active inflammation due to refluxesophagitis. Results for the Mesoscale IL-8 measurements were verifiedusing an IL-8 ELISA (data not shown).

Time course for detection of IL-8 protein in the esophagus using the ESTin a patient with GERD. In order to establish a time course for thedetection of esophageal inflammation by the EST, two ESTs were performedon different days in the same patient with well-defined GERD. Thestrings were removed after 1 or 12 hours, the esophageal segmentsidentified using pH assessments as above, and cut into proximal anddistal halves. The adherent secretions were eluted and assayed byMesoscale for IL-8 protein content (FIG. 13). IL-8 was clearlydetectable in both the proximal and distal esophagus after the stringwas in the esophagus for 1 hour, but IL-8 levels were ˜3-fold higherwhen the string was left in place for 12 hours (FIG. 13). These resultsshow that the EST is capable of detecting the presence of inflammatorycytokines in situ in at little as 1 hour, with increased levels detectedafter longer EST periods.

Increased IL-8 compared to other inflammatory cytokines as determined byEST in a GERD patient. EST was performed in a well-defined patient withGERD and the string removed after 12 hours. The esophageal stringsegment (based on pH probe) was cut into proximal and distal halves, theadherent secretions eluted with sample buffer, and the secretionsassayed by Mesoscale for pro-inflammatory cytokines as above. Highlevels of IL-8 were measured in the proximal and distal samples from theEST relative to other cytokines including IL-2, IL-4, IL-10, IL-13, andTNF-α that were below detectable limits of the assay (FIG. 14). IL-1β,IL-5 and INF-γ were also detectable, but at much lower levels than IL-8(FIG. 14).

Detection of IL-8 mRNA by EST in the esophagus of a patient with GERD.The same GERD patient as above performed a 1 hour EST and total RNA wasextracted from the EST utilizing TRIZOL extraction techniquesimmediately after harvesting the string. Quantitative RT-Q-PCR wasperformed utilizing primers specific for IL-8 and normalized to theexpression of β-actin determined using β-actin-specific primers (FIG.15). Results show that the EST detected IL-8 mRNA in the both proximaland distal esophageal samples, consonant with previous reports of IL-8expression in tissue sections in GERD, and importantly, support thetechnical ability of the EST to retain cells from the esophageal lumenfor detection of inflammatory cell biomarker mRNAs.

Detection of decreased IL-8 levels by EST in a patient with GERDfollowing PPI treatment. The EST was performed in a well-defined patientwith GERD before and after treatment for one month with a proton pumpinhibitor (PPI). For both ESTs, the string was removed after 1 hour, theesophageal segment cut into proximal and distal halves, the luminalsecretions eluted and assayed by Mesoscale for IL-8 protein, and resultsexpressed as pg/ml IL-8/cm eluted string (FIG. 16). The EST-detectedlevels of IL-8 protein decreased by ˜32% and 55% compared to thepre-treatment samples for measurements in the proximal and distalesophagus, respectively, demonstrating the feasibility for using the ESTto monitor treatment-induced changes in biomarkers of esophagealinflammation, as proposed for studies of the effects of nutritional andcorticosteroid treatments on esophageal inflammation in patients withEE.

Taken together, these preliminary results support a role for the EST inidentifying and quantitating biomarkers of esophageal inflammation atboth the protein and mRNA levels in an anatomically and cytokinespecific fashion. In addition, they demonstrate how the EST can be usedto follow treatment-induced changes in inflammatory responses within theesophageal lumen.

Experimental Design and Methods

Another unmet need in the assessment and evaluation of patients with EEis that except for endoscopy with biopsy, no other method exists as ameans for assessing adequacy of responses to treatment. Symptoms,blood/stool tests and endoscopic appearance do not necessarily correlatewith the histological assessments of esophageal inflammation, and aminimally invasive tool is urgently necessary to aid in determining EE'snatural history and treatment effectiveness. As such, the esophagealinflammatory milieu may be investigated with the EST before and aftertreatment (nutritional elimination diets or corticosteroid).

Eosinophil derived granule proteins were identified as primary targetsin Example 3. While a body of literature suggests that IgE plays a rolein the pathogenesis of EE, no prospective studies have yet determinedIgE-associated markers in luminal secretions of patients with EE. Thus,CD23, FcERI, and mast cell tryptase are measured to characterize themucosa (immunohistochemical staining) and luminal secretions (Westernblot analysis). CD23 is a cell surface molecule that has recently beenreported capable of mediating epithelial transcytosis of IgE/allergencomplexes for presentation and induction of mast cell activation (Li etal., 2006). This has been previously measured in stool effluents frompatients with food allergic diseases. FcERI is the high affinityreceptor for IgE that is typically associated with mast cells andbasophils (Fiebiger et al., 2005; Sabroe et al., 2002). While it has notbeen previously measured in intestinal secretions, preliminary evidenceshows that it is increased in esophageal tissues in association with EE(unpublished data).

In this light, it is expected that the presence of these targets may bemeasured by EST in esophageal secretions. Tryptase is a mastcell/basophil product that provides clinical evidence of degranulation.Since mast cells are increased in the esophageal tissues affected by EE,it is expected that levels of tryptase will concordantly be increased inesophageal secretions and can be detected by EST. Second, previousstudies support the hypothesis that EE is a Th2-mediated disease (Mishraet al., 2001; Mishra et al., 2000; Mishra et al., 2007). Affectedtissues have eosinophils, increased IL-5 expression, and murine modelssupport an IL-5 dependent response. Treatment of limited number of EEpatients with anti-IL-5 (Mepolizumab™) have shown therapeutic responsesthat included up to a 50% decrease in the numbers of esophagealeosinophils (Stein et al., 2006). In contrast, some patients expressincreased TNF-α and do not respond to food elimination diets. As such,the definition of the inflammatory milieu will be important to furthercharacterize the disease state and determine potential therapeutictargets.

To date, very little research has comprehensively examined theesophageal milieu associated with EE (Blanchard et al., 2006a; Straumannet al., 2001; Konikoff et al., 2006; Gupta et al., 2006a; Gupta et al.,2006b). One study determined that measurements of absolute peripheralblood eosinophil counts, plasma EDN and eotaxin-3 levels were associatedwith esophageal inflammation. Thus, the EST will be used to measure theabove-noted inflammatory mediators in samples from theplasma-(peripheral site), mucosal tissue-(gold standard), EST (localassessment) to determine whether associations exist. Furthermore, thesepanels of inflammatory mediators measured by the EST before and aftertreatment will be compared to determine their usefulness in monitoringdisease. Also, it is expected that comparisons of EST levels (pre- andpost-treatment) will begin to identify potential inflammatory moleculesimpacted by designated treatments that may serve as diagnostic cluesand/or biomarkers.

1. Measure Plasma, Esophageal Mucosal (Biopsy) and Luminal (EST)Quantities of Eosinophilic Inflammation, Mediators of IgE Inflammation,and Th1/Th2 Cytokines Pre- and Post EE Treatment.

Evaluation of EST. Inflammatory mediators associated with EE will bequantified from samples derived from three sources, plasma, tissue, andesophageal lumen before and after treatment. Samples will be obtainedfrom EE patients and normal controls as in Example 3. Each sample willundergo the following analyses (method of analysis);

1. Blood/plasma—eosinophil count, eosinophil derived granule proteins(ELISA), CD23 (ELISA), FcεRI (ELISA), tryptase (ELISA), eotaxin-3(ELISA), and Th1 [IL-2, TNF-α, IFN-γ] and Th2 [L-4, IL-5 and IL-13]cytokines (Mesoscale).

2. Mucosal biopsy—eosinophil count, eosinophil derived granule proteins(immunohistochemistry-IHC), CD23 (IHC), FcεRI (IHC), tryptase (IHC),eotaxin-3 (mRNA), and Th1 [IL-2, TNF-α, IFN-γ] and Th2 [L-4, IL-5 andIL-13] cytokines-(mRNA).

3. EST—eosinophil derived granule proteins (ELISA), CD23 (ELISA), FcεRI(ELISA), tryptase (ELISA), eotaxin-3 (ELISA), and Th1 (IL-2, TNF-α,IFN-γ) and Th2 (IL-4, IL-5 and IL-13) cytokines (Mesoscale).

Samples will be collected before and after 8-12 weeks of eithernutritional elimination (dietary elimination of specific foods asdetermined by skin prick testing or elemental diet) or corticosteroidtreatments (systemic or topical [inhaled/swallowed] corticosteroids)(Ngo and Furuta, 2005). Both of these treatments have been shown toprovide effective and durable remission of clinical and histologicalfeatures of EE when used for 4-12 weeks. As a part of the standard ofcare, an endoscopy will be performed at the end of treatment. ESTs willbe provided to the patient at the end of treatment to be swallowed thenight before the endoscopy.

a. Protein Analyses

ELISA—Most targets have commercially available ELISAs (Furuta et al.,2000; Furuta et al., 2001; Karhausen et al., 2004). Sources of ELISA orantibodies are noted here—CD23 (BD Bioscience), FcERI (ELISA),Eotaxin-1, 2, 3(R&D Systems), Tryptase (R&D Systems).

Mesoscale—Mesoscale™ technology (Gowan et al., 2007) is a multiplexquantitative system for analyzing up to 12 target proteins in a singlewell of a 96-well plate. This technology needs only 2 μl of sample/welland the 96-well format allows for high throughput sampling and analysis.Data are provided in a quantitative format as pg protein per ml. For thestudies, standardized Th1/Th2 templates will be used to assess thepresence of Th1 (IL-2, TNF-α, IFN-γ) and Th2 (IL-4, IL-5 and IL-13)cytokines.

Immunohistochemistry—Tissue sections will undergo immunohistochemicalanalysis with specific antibodies as described above; antibodies forELISA are also available for immunohistochemical analysis as previouslydescribed (Walsh et al., 1999; Desai et al., 2005; Teitelbaum et al.,2002). Briefly, all specimens will be formalin-fixed, paraffin-embedded,cut serially and either stained with hematoxylin and eosin or used forimmunohistochemical studies. For immunohistochemical staining, sectionswill be dewaxed, rehydrated, peroxidase activity quenched, blocked andincubated with primary antibody and then secondary antibody. Colorreaction will be then developed using the diaminobenzidine method andcounterstained with hematoxylin, dehydrated, and mounted. Appropriatepositive and negative controls will be included in each stainingreaction. Quantification of positively stained cells for each of theselected antibodies will be performed.

b. RNA Analyses

Tissues will be homogenized with TRIZOL reagent to isolate total RNAthat will then undergo reverse transcription and Q-PCR analysis using aBIORAD Real-Time iCycler PCR instrument. Primers for Th1 (IL-2, TNF-α,IFN-γ) and Th2 (IL-4, IL-5 and IL-13) cytokines have been purchased fromInvitrogen.

c. Statistical Analyses

Linear regression will be used to model the percent change in quantityof histological eosinophilic inflammation with the change in markerlevel. Multiple linear regression will be used to assess therelationship of multiple markers with the pathological outcomesimultaneously.

2. Determine Associations of Pro-Inflammatory Milieus as Measured by ESTwith Clinical State.

Pre- and post-treatment inflammatory markers levels as determined by ESTwill be compared with the patient's primary symptoms. Patients' symptomswill be recorded before and after treatment as improved, the same andworse.

Statistical Analyses. Chi-square test will be used to test theassociation of change in symptom (improved, same, worse) and change inbiomarker (increased, same, decreased). Proportion odds model will beused to examine the association of multiple markers with the clinicalstatus simultaneously. Separate analysis will be done for EE patientsand those with normal histology.

All of the methods and apparatus disclosed and claimed herein can bemade and executed without undue experimentation in light of the presentdisclosure. While the compositions and methods of this invention havebeen described in terms of particular embodiments, it will be apparentto those of skill in the art that variations may be applied to themethods and apparatus and in the steps or in the sequence of steps ofthe method described herein without departing from the concept, spiritand scope of the invention. More specifically, it will be apparent thatcertain agents which are both chemically and physiologically related maybe substituted for the agents described herein while the same or similarresults would be achieved. All such similar substitutes andmodifications apparent to those skilled in the art are deemed to bewithin the spirit, scope and concept of the invention as defined by theappended claims.

REFERENCES

The following references, to the extent that they provide exemplaryprocedural or other details supplementary to those set forth herein, arespecifically incorporated herein by reference.

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1. A method for diagnosing a disease of the esophagus comprising: (a)deploying a device into the esophagus of a subject; (b) removing thedevice after a predetermined period of time; (c) analyzing the devicefor a diagnostic indicator of a disease of the esophagus; and (d)evaluating the diagnostic indicator to diagnose a disease of theesophagus.
 2. The method of claim 1, wherein the disease isgastroesophageal reflux disease (GERD).
 3. The method of claim 2,wherein the diagnostic indicator is IL-8 mRNA or IL-8 protein.
 4. Themethod of claim 3, wherein the IL-8 is IL-8 mRNA.
 5. The method of claim3, wherein the IL-8 is IL-8 protein.
 6. The method of claim 3, whereinthe predetermined time is between 15 minutes and 12 hours.
 7. The methodof claim 6, wherein the predetermined time is 15 minutes.
 8. The methodof claim 6, wherein the predetermined time is 1, 2, 3, 4, 5, 6, 7, 8, 9,10, 11, 12, 18, or 24 hours.
 9. The method of claim 8, wherein thepredetermined period of time is 12 hours.
 10. The method of claim 8,wherein the predetermined period of time is 1 hour.
 11. The method ofclaim 1, wherein the disease is Eosinophilic Esophagitis (EE).
 12. Themethod of claim 11, wherein the diagnostic indicator is an eosinophilgranule protein.
 13. The method of claim 12, wherein the eosinophilgranule protein is a major basic protein (MBP), an eosinophil cationicprotein (ECP), an eosinophil peroxidase (EPO), or an eosinophil-derivedneurotoxin (EDN).
 14. The method of claim 13, wherein the eosinophilgranule protein is a major basic protein.
 15. The method of claim 13,wherein the major basic protein is major basic protein 1 (MBP1).
 16. Themethod of claim 12, wherein the eosinophil granule protein is IL-5induced.
 17. The method of claim 16, wherein the IL-5 induced eosinophilgranule protein is EPO, MBP1, or CLC/Gal-10.
 18. The method of claim 11,wherein the diagnostic indicator is selected from the group consistingof a cytokine and a chemokine.
 19. The method of claim 18, wherein thechemokine is an eotaxin.
 20. The method of claim 19, wherein the eotaxinis eotaxin-3.
 21. The method of claim 11, wherein the diagnosticindicator is a cellular infiltrate or pH.
 22. The method of claim 11,wherein the diagnostic indicator is a marker of an allergic response.23. The method of claim 22, wherein the marker of an allergic responseis an IgE, a tryptase, CD23, FcR or an allergen.
 24. The method of claim11, wherein the diagnostic indicator comprises at least two of the groupconsisting of an eosinophil granule protein, a cytokine, a chemokine, acellular infiltrate, and a marker of an allergic response.
 25. Themethod of claim 24, wherein the predetermined time is between 15 minutesand 12 hours.
 26. The method of claim 26, wherein the predetermined timeis 15 minutes.
 27. The method of claim 25, wherein the predeterminedtime is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 18, or 24 hours.
 28. Themethod of claim 27, wherein the predetermined period of time is 12hours.
 29. The method of claim 27, wherein the predetermined period oftime is 1 hour.
 30. The method of claim 1, further comprisingquantifying the diagnostic indicator.
 31. The method of claim 30,wherein quantifying the diagnostic indicator is performed by ELISA orMesoscale.
 32. The method of claim 1, wherein the device comprises apharmaceutical capsule having an opening, a malleable drag materialwithin the capsule, and a line embedded in the drag material and runningthrough the opening of the capsule.
 33. The method of claim 32, whereinthe opening is a perforated opening.
 34. The method of claim 32, whereinthe pharmaceutical capsule is dissolvable.
 35. The method of claim 32,wherein the line comprises a distal segment and a proximal segment. 36.The method of claim 35, wherein the proximal segment is made of string.37. The method of claim 36, wherein the string comprises an absorbentfiber.
 38. The method of claim 36, wherein the string comprises atextured fiber.
 39. The method of claim 1, wherein the device furthercomprises a capture agent for one or more diagnostic indicators of adisease of the esophagus.
 40. A method for assessing a treatment of adisease of the esophagus comprising: (a) deploying a device into theesophagus of a subject; (b) removing the device after a predeterminedperiod of time; (c) analyzing the device for a diagnostic indicator of adisease of the esophagus; and (d) evaluating the diagnostic indicator toassess the treatment of a disease of the esophagus.
 41. The method ofclaim 40, wherein the disease is Eosinophilic Esophagitis (EE) orgastroesophageal reflux disease (GERD).
 42. A method for measuringesophageal inflammation comprising: (a) deploying a device into theesophagus of a subject; (b) removing the device after a predeterminedperiod of time; (c) analyzing the device for a diagnostic indicator ofesophageal inflammation; and (d) evaluating the diagnostic indicator tomeasure esophageal inflammation.